ENZYMATIC MOLECULE MIMICKING ANTI-OXIDANT ACTIVITY
20210244635 · 2021-08-12
Assignee
Inventors
Cpc classification
A61Q17/04
HUMAN NECESSITIES
A61K8/0241
HUMAN NECESSITIES
International classification
A61Q17/04
HUMAN NECESSITIES
Abstract
Semiconductor colloids grafted covalently with an antioxidant, the colloids being semiconductor colloids constituted by at least one element chosen from the group comprising C, Si, Ge, Sn, S, Se, Te, B, N, P, As, Al, Sb, Ga, In, Cd, Zn, O, Cu, Cl, Pb, Tl, Bi, Ti, U, Ba, Sr, Li, Nb, La, I, Mo, Mn, Ca, Fe, Ni, Eu, Cr, Br, Ag, Pt, Hg, and combinations thereof.
Claims
1. A semiconductor colloids grafted covalently with an antioxidant, the colloids being semiconductor colloids constituted by at least one element chosen from the group consisting of C, Si, Ge, Sn, S, Se, Te, B, N, P, As, Al, Sb, Ga, In, Cd, Zn, O, Cu, Cl, Pb, Tl, Bi, Ti, U, Ba, Sr, Li, Nb, La, I, Mo, Mn, Ca, Fe, Ni, Eu, Cr, Br, Ag, Pt, Hg, and combinations thereof.
2. The colloids according to claim 1, wherein the semiconductor colloids are colloids of zinc oxide, ZnO, or colloids of rutile titanium dioxide, TiO.sub.2.
3. The colloids according to claim 1, wherein the antioxidant is a phenolic aldehyde or a phenolic acid.
4. The colloids according to claim 1, wherein the antioxidant is chosen from the group consisting of 2-hydroxybenzaldehyde, 3-hydroxybenzaldehyde, 4-hydroxybenzaldehyde, and 3,4-dihydroxybenzaldehyde.
5. The colloids according to claim 4, wherein the antioxidant is 3-hydroxybenzaldehyde.
6. The colloids according to claim 4, wherein the antioxidant is 3,4 dihydroxybenzaldehyde.
7. The colloids according to claim 1, wherein the covalent grafting is in the form of a spacer arm positioned between the semiconductor colloid and the antioxidant, the spacer arm comprising in the range 1 to 8 carbons, and having an alkoxysilane function capable of binding itself covalently to the colloid and a function of the hydroxyl type, of the phosphate type, or of the amine type capable of binding itself to the antioxidant.
8. The colloids according to claim 7, wherein the spacer arm is 3-(Aminopropyl)triethoxysilane.
9. The colloids according to claim 1, wherein the antioxidant is chosen from the group consisting of the compounds or their derivatives designated by the following INCI names: octadecyl di-t-butyl-4-hydroxyhydrocinnamate; pentaerythrityl tetra-di-t-butyl hydroxyhydrocinnamate; 2,6-Bis(1,1-dimethylethyl)-4-methylphenol; bis-ethylhexyl hydroxydimethoxy benzylmalonate; manganese dioxide; colloidal platinum; tert-butylhydroquinone; tetrabutyl ethylidenebisphenol; sodium bisulfate; sodium metabisulfite; thioglycolic acid; thiotaurine; thioctic acid; dilauryl thiodipropionate; aminoethanesulfinic acid; triethyl citrate; sodium erythorbate; sorbityl furfural; erythorbic acid; perillyl alcohol; pyridyloxide t-butylnitrone; ergothioneine; melatonin; acetyl cysteine; cysteine; lysine hydrochloride; carnosic acid; tyrosyl histidine HCl; histidine hydrochloride; pyridoxine serinate; superoxide dismutase; aminopropyl ascorbyl phosphate; ascorbic acid; ascorbic acid polypeptide; ascorbyl dipalmitate; ascorbyl glucoside; ascorbyl linoleate; ascorbyl methylsilanol pectinate; ascorbyl palmitate; ascorbyl tetraisopalmitate; ascorbyl tocopheryl maleate; trisodium ascorbyl palmitate phosphate; disodium ascorbyl sulfate; calcium ascorbate; methylsilanol ascorbate; sodium ascorbate; sodium ascorbyl phosphate; sodium ascorbyl/cholesteryl phosphate; tetrahexyldecyl ascorbate; magnesium ascorbyl phosphate; tocopherol; tocopheryl acetate; tocopheryl linoleate; tocopheryl linoleate/oleate; tocopheryl nicotinate; tocopheryl retinoate; sodium tocopheryl phosphate; dioleyl tocopheryl methylsilanol; potassium ascorbyl tocopheryl phosphate; dodecyl gallate; epigallocatechin gallate EGCG; propyl gallate; ethyl ferulate; ethylhexyl ferulate; chitosan ascorbate; chitosan glycolate; apigenin; tiliroside; alpha-arbutin; arbutin; baicalin; quercetin; quercetin caprylate; isoquercitrin; diethylhexyl syringylidenemalonate; dihydroxy methylchromone; dimethoxy di-p-cresol; dimethylmethoxy chromanol; ethylbisiminomethylguaiacol manganese chloride; hesperidin methyl chalcone; kojic acid; kojic dipalmitate; madecassoside; asiaticoside; magnolol (5,5′-diallyl-2,2′-dihydroxybiphenyl); nordihydroguaiaretic acid; phenylethyl resorcinol; resveratrol; troxerutin (3′,4′,7-tris(hydroxyethyl)rutin); glucosylrutin; rutin (4H-1-benzopyran-4-one); disodium rutinyl disulfate; tetrahydrobisdemethoxydiferuloylmethane; tetrahydrodemethoxydiferuloylmethane; tetrahydrodiferuloylmethane; tococysteamide; totarol; hydroxydecyl ubiquinone; ubiquinone; carotenoids; lycopene, gallic acid; and caffeic acid.
10. A topical composition comprising the colloids according to claim 1.
11. A topical composition comprising the colloids according to claim 19.
12. A topical composition according to claim 10, wherein it is devoid of any sun filters that are hydrophilic and/or lipophilic, inorganic and/or organic.
13. A topical composition according to claim 10, wherein it is in aqueous form, micellar form, or Pickering emulsion form.
14. A food supplement including colloids according to claim 1.
15. The colloids according to claim 2, wherein the antioxidant is a phenolic aldehyde or a phenolic acid.
16. The colloids according to claim 15, wherein the antioxidant is chosen from the group consisting of 2-hydroxybenzaldehyde, 3-hydroxybenzaldehyde, 4-hydroxybenzaldehyde, and 3,4-dihydroxybenzaldehyde.
17. The colloids according to claim 16, wherein the antioxidant is 3-hydroxybenzaldehyde.
18. The colloids according to claim 16, wherein the antioxidant is 3,4 dihydroxybenzaldehyde.
19. The colloids according to claim 16, wherein the covalent grafting is in the form of a spacer arm positioned between the semiconductor colloid and the antioxidant, the spacer arm comprising in the range 2 to 4 carbons, and having an alkoxysilane function capable of binding itself covalently to the colloid and a function of the hydroxyl type, of the phosphate type, or of the amine type capable of binding itself to the antioxidant.
20. A topical composition comprising the colloids according to claim 16, wherein the antioxidant is 3-hydroxybenzaldehyde or 3,4 dihydroxybenzaldehyde, and wherein the covalent grafting is in the form of a spacer arm positioned between the semiconductor colloid and the antioxidant, the spacer arm comprising in the range 2 to 4 carbons, and having an alkoxysilane function capable of binding itself covalently to the colloid and a function of the hydroxyl type, of the phosphate type, or of the amine type capable of binding itself to the antioxidant.
Description
DESCRIPTION OF THE FIGURES
[0082]
[0083]
[0084]
[0085]
[0086]
[0087]
EXAMPLES OF IMPLEMENTATIONS OF THE INVENTION
Example 1: ZnO/3-hydroxybenzaldehyde
1/ Synthesis of the Colloids
[0088] The percentages are given by weight of the composition.
[0089] A zinc oxide precursor, such as anhydrous zinc acetate (2%) and sodium hydroxide (1.5%), was mixed with a solvent or with a mixture of solvents comprising ethanol (85%) and diethylene glycol (8%) and sealed in an autoclave. The solvent may also be benzyl alcohol, phenol, oleyl alcohol, butanol, propanol, isopropanol, water, tetrahydrofuran, ethanol, methanol, acetonitrile, toluene, PGMEA, PGPE, PGME, 2-methyl-1-propanol, or triethylene glycol monomethyl ether.
[0090] The reaction medium was placed under mechanical agitation at 60° C. for about 30 minutes, until the salts had dissolved. Water (2%) was then added. Cloudiness was observed that marked the start of formation of the particles. The reaction was kept at 65° C. for one and a half hours.
[0091] Zinc oxide nanocrystals that were spherical and 7 nm in diameter were collected.
2/ Preparation of the Colloids Col/AntiOx
[0092] Colloids of the invention were prepared using the zinc oxide (ZnO) colloids obtained above and a precursor or spacer arm of the antioxidant 3-hydroxybenzaldehyde. Particles of ZnO/3-hydroxybenzaldehyde were thus obtained. In practice, the nanocrystals formed were functionalized in situ with (3-Aminopropyl)triethoxysilane (0.5%). On addition, the solution cleared slightly. This functionalization was performed for an additional 3 hours. Finally the antioxidant 3-Hydroxybenzaldehyde (1.5%) was added. The reaction was maintained for 9 hours.
[0093] The reactor was then cooled to ambient temperature. The particles were collected and centrifuged at 3000 rpm for 15 minutes. They were then washed in ethanol, and then centrifuged again. Finally, the particles were dispersed to the desired concentration in water (mass concentration in the range 10% to 15%).
[0094] The small size of the ZnO particles made it possible to have a larger area to cover and thus to graft a larger number of antioxidant molecules to the surface of each of the particles.
[0095] The diameter of the ZnO colloids was measured by means of an X-ray diffractometer (XRD). The wavelength produced by the diffractometer corresponded to the Cu-Kα line equal to 1.54 Å. The other parameters used corresponded to an acceleration voltage of 40 kV, to an electric current of 40 mA, and to a Bragg-Brentano geometry.
[0096] The X-ray diffraction patterns were measured on powder with an XRD of Cu-Kα source in transmission. The X-ray diffraction pattern of colloids before functionalization (grafting) with the antioxidant is shown in
Example 2: TiO.SUB.2./3-hydroxybenzaldehyde
[0097] Under the same conditions as in the preceding example, the same antioxidant was grafted onto rutile titanium dioxide. 5 g of rutile titanium dioxide was dispersed in a mixture of 950 mL ethanol and 50 mL diethylene glycol. The mixture was heated to 65° C. and agitated. 5 mL of water was added, and then 8 mL of (3-Aminopropyl)triethoxysilane. The mixture was left to be agitated for two hours, and then 5 g of 3-hydroxybenzaldehyde was added, and the mixture was left to be agitated and heated at 65° C. for 9 hours. The mixture was then cooled to ambient temperature. The particles were collected and centrifuged at 3000 rpm for 15 minutes. They were then washed in ethanol, and then centrifuged again. Finally, the particles were dispersed to the desired concentration in water.
[0098] Similar syntheses may be performed by implementing as 2-hydroxybenzaldehyde or 4-hydroxybenzaldehyde as antioxidant.
Example 3: TiO.SUB.2./3,4 dihydroxybenzaldehyde
[0099] Under the same conditions as in the preceding example, the 3,4 dihydroxybenzaldehyde was grafted onto rutile titanium dioxide. 5 g of rutile titanium dioxide was dispersed in a mixture of 950 mL ethanol and 50 mL diethylene glycol. The mixture was heated to 65° C. and agitated. 5 mL of water was added, and then 8 mL of (3-Aminopropyl)triethoxysilane. The mixture was left to be agitated for two hours, and then 5 g of 3,4 dihydroxybenzaldehyde was added, and the mixture was left to be agitated and heated at 65° C. for 9 hours. The mixture was then cooled to ambient temperature. The particles were collected and centrifuged at 3000 rpm for 15 minutes. They were then washed in ethanol, and then centrifuged again. Finally, the particles were dispersed to the desired concentration in water.
Example 4: Comparison of the Kinetic Time of the Antioxidant Activity of the Colloids Col/AntiOx (ZnO/3-hydroxybenzaldehyde) with that of a Free Antioxidant (ZnO/3-hydroxybenzaldehyde)
[0100] The rate of action of the colloids Col/AntiOx was estimated by measuring the decomposition kinetics of 2,2-diphenyl-1-picrylhydrazyl (DPPH).
[0101] DPPH is a molecule that keeps its free radical capacity stably. This radical species absorbs light at 520 nm (purple color of the solution) and becomes colorless or pale yellow after neutralization by an antioxidant. It is thus possible to monitor the neutralization reaction by measuring the intensity of the measurement of absorption of the radical DPPH as a function of time.
[0102] For that purpose, two solutions in ethanol were prepared as follows: [0103] a control solution containing DPPH at a [DPPH].sub.0 concentration of 0.1 mol/L; and [0104] a test solution containing DPPH at a [DPPH].sub.0 concentration of 0.1 mol/L and an antioxidant at a concentration such that 90% of the DPPH was consumed after 2 hours (as determined based on the CI.sub.50 measurements).
[0105] Absorbance was measured by means of a UV/vis/NIR spectrometer. 2.5 mL of solution was poured into a polystyrene vessel (optical path=1 cm). The UV/visible absorption spectrum from 310 nm to 700 mm was measured for 300 seconds. The value of the absorbance at 520 nm made it possible to determine the concentration of radical DPPH at a given time using the Beer-Lambert equation:
A.sub.520 nm(τ)=ε.sub.DPPH.Math.l.Math.[DPPH].sub.τ
[0106] In accordance with that equation, A corresponds to the measured absorbance, 8.sub.DPPH corresponds to the mass coefficient of DPPH, l (cm) corresponds to the optical path through the sample, and [DPPH].sub.τ (g/L) corresponds to the mass concentration of the sample.
[0107] The curves obtained were honed using a kinetic model that made it possible to go back to the reaction rate or “kinetic” constant K and the half-life constant z of the reaction.
[0108] The graph showing the rate of action of the colloids functionalized with an antioxidant (ZnO/3-hydroxybenzaldehyde) and the rate of action of the free, i.e. not grafted, antioxidant (3-hydroxybenzaldehyde) is shown in
[0109] The results show that the ZnO/3-hydroxybenzaldehyde antioxidant reacts more quickly with DPPH than the free 3-hydroxybenzaldehyde.
Example 5: Assessing the Regeneration of the Antioxidant Activity of the Col/AntiOx Colloids
5.1/ Assessment Over Time
[0110] A stock solution of ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic) acid) at 8 mM was incubated, at equal volume, with a solution at 3 mM comprising metmyoglobin and hydrogen peroxide so as to produce an ABTS radical cation. The solution obtained was diluted with phosphate buffer (0.2 M, and pH 7.4) containing 150 mM of NaCl so as to obtain an absorbance of 1.5 at 734 nm.
[0111] Samples of 30 μL of ZnO/3-hydroxybenzaldehyde (example 2/) dispersed at 240 g/L in water were added to 2970 μL of the 0.07 mM ABTS cation solution in water, and then placed under agitation in the dark.
[0112] After 30 minutes of incubation, the ABTS was totally degraded.
[0113] The solution obtained after this step of degradation of the entire ABTS substrate was separated: a fraction of the solution being placed in the dark, and the remaining fraction being placed under UV irradiation. After 30 minutes of exposure (in the dark or under UV), 30 μL of a concentrated solution of ABTS (7 mM) was added, and then the solutions were, once again, placed in the dark under agitation for 5 hours. The absorption was then measured every 30 minutes.
[0114] The data is shown in
[0115] The results show that the solution containing ZnO/3-hydroxybenzaldehyde colloids and exposed to UV radiation has an ABTS degradation capacity that is considerably greater than the solution containing ZnO/3-hydroxybenzaldehyde colloids that remained in the dark. The ABTS degradation activity observed for the solution placed in the dark corresponds to the residual anti-radical activity inherent to the ZnO/3-hydroxybenzaldehyde complex. Thus, after 120 minutes, 65% of the ABTS was degraded by the solution that had been exposed to UV radiation, as compared with 18% for the solution that remained in the dark.
[0116] In conclusion, the ZnO/3-hydroxybenzaldehyde colloids of the invention have the capacity to regenerate their antioxidant activity when they are exposed to UV radiation.
5.2/ Comparison with Tocopherol.
[0117] The protocol for preparing the solutions as in point 4.1/ was repeated with a concentration of ZnO/3-hydroxybenzaldehyde colloids of 0.5 g/L and a concentration of tocopherol of 1.5 g/L.
[0118] The solutions obtained were separated: a fraction of the solution being placed in the dark, and the remaining fraction being placed under UV irradiation. After 30 minutes of exposure (to the dark or to UV), 30 μL of a concentrated solution of ABTS (7 mM) was added, and the absorption was then measured.
[0119] The data is shown in
[0120] The results show that the solution containing tocopherol had the same ABTS degradation capacity (22%) after exposure to UV or after exposure to the dark.
[0121] The solution containing ZnO/3-hydroxybenzaldehyde colloids and exposed to darkness had an ABTS degradation capacity that was very considerably greater than the tocopherol (76%). This activity was further increased after exposure to UV (89%).
[0122] In conclusion, the ZnO/3-hydroxybenzaldehyde colloids of the invention have an anti-radical activity that is more effective than a conventional antioxidant and a capacity to regenerate their antioxidant activity when they are exposed to UV radiation.
Example 6: Assessing the Antioxidant Activity of the Colloids Functionalized with Antioxidants (ZnO/3-hydroxybenzaldehyde) of the Invention
[0123] The antioxidant activity of the ZnO/3-hydroxybenzaldehyde colloids was estimated by measuring the decomposition of 2,2-diphenyl-1-picrylhydrazyl (DPPH). The results were expressed in CI.sub.50 and/or in mg or μmol equivalent of the selected antioxidant sample. This method shows the capacity of a molecule to trap free radicals by transfer of electrons and/or of protons resulting from oxidation phenomenon. The protocol was as follows:
[0124] an ethanol DPPH solution was prepared at 0.1 mM; [0125] the samples (antioxidant) to be tested were prepared at various masses lying in the range 4 mg to 40 mg; [0126] in a PMMA (poly(methyl methacrylate)) vessel, 3900 μL of solution of DPPH was mixed with 100 μL of sample to be tested. The reaction took place under incubation at ambient temperature, for 2 hours, and then absorbance at 515 nm was measured; and [0127] the percentage of antioxidant activity was plotted as a function of the concentration of the sample in question (in μM).
[0128] The percentage of antioxidant activity was determined using the following formula:
% antioxidant activity=(A.sub.samp/A.sub.blank×100
[0129] where A.sub.samp corresponds to the absorbance of the sample whereas A.sub.blank corresponds to the absorbance of a solution without any sample (100 μL of ethanol+3900 μL of a DPPH/ethanol solution).
[0130] The curve obtained has a linear portion and an asymptotic portion. The linear zone is in the form % AO=χ×[sample].
[0131] This formula makes it possible to determine the sample concentration that makes it possible to trap 50% of the radicals present, i.e. to determine the CI.sub.50 in g/L (minimum concentration that is inhibiting at 50%) of the sample in question:
CI.sub.50=50/χ
[0132] where x represents the slope of the linear regression line.
[0133] The antioxidant activity of the colloids of example 2 (ZnO/3-hydroxybenzaldehyde) was compared with that of conventional antioxidants listed below:
[0134] Sample of 30 μL diluted to 0.5 g/L was placed in 2970 L of a solution at 0.07 mM of ABTS; [0135] Green tea extract containing over 40% of epigallocatechin gallate (EGCG); [0136] 87.3% of α-tocopherol+12.7% of soybean oil; [0137] 100% of L-ascorbic acid (Vitamin C); and [0138] 100% of propyl gallate.
[0139] The results are shown in
[0140]
[0141] Furthermore, an antioxidant activity greater than that of the other antioxidants at a concentration less than or equal to 0.15 g/L and similar at a concentration greater than 0.15 g/L was observed.
[0142] The antioxidant activity of ZnO/3-hydroxybenzaldehyde was thus greater than those of the other antioxidants tested.
Example 7: Assessing the Protective Power of the Composition of the Invention Against Generation of Primary and Secondary Free Radicals
[0143] The trial was performed by the “spin trapping” method coupled with Electron Paramagnetic Resonance (EPR) for conducting trapping experiments in competition by using DIPPMPO (5-(Diisopropoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide) as the trapping molecule. This compound is used conventionally for detecting and studying the superoxide radical using this technique.
[0144] The variation in the intensity of the signal of the adduct DIPPMPO-OOH was monitored as a function of time in the presence and in the absence of the creams. The method was assessed by studying the decomposition kinetics of a model radical (TEMPOL or 1-Oxyl-2,2,6,6-tetramethyl-4-hydroxypiperidine) in the presence and in the absence of the creams. Over the duration of the trial, i.e. 20 minutes, no loss of signal from the TEMPOL was observed for the 4 experiments (control+3 samples). Also, control analyses in the presence of Superoxide Dismutase (SOD) were conducted.
TABLE-US-00001 TABLE 2 Percentage of inhibition of the DIPPMPO-OOH signal as a function of time. Inhibition Inhibition Inhibition Inhibition at 3 at 5 at 10 at 15 Cream minutes minutes minutes minutes Sample A Complex of the 100% 100% 100% 100% invention (ZnO/3- hydroxybenzaldehyde) Sample B Mixture of 48% 50% 45% 53% antioxidants: squalane + tocopherol acetate + magnesium ascorbyl phosphate Sample C Mixture of 20% 23% 37% 44% antioxidants: tocopherol acetate + tocopherol + ergothioneine
[0145] The values indicated in the table are the means of 3 identical experiments (the fluctuations being less than 10%).
[0146] The inhibition kinetics are shown in
[0147] Different superoxide radical trapping properties were observed for each sample.
[0148] Sample B showed a reduction of 48% at 3 minutes (53% at 5 minutes) in the signal of the DIPPMPO-OOH adduct, suggesting inhibition of half of the oxidizing activity.
[0149] Sample C showed a reduction of 20% at 3 minutes (44% at 5 minutes) in the signal of the DIPPMPO-OOH adduct, suggesting inhibition that was less effective than the inhibition of the sample B.
[0150] For the complex of the invention, i.e. sample A (the ZnO/3-hydroxybenzaldehyde colloids), no signal corresponding to the DIPPMPO-OOH adduct was observed. Thus, all of the superoxide radical produced was trapped.
[0151] During these experiments, formation of an additional radical was observed (except for the sample of the invention). According to the Applicant, it appeared that that new radical came from a Haber Weiss reaction or a Fenton reaction involving the presence of transition metal salts available for reacting with the superoxide radical or with the hydrogen peroxide (generated by spontaneous dismutation of the superoxide radical). Thus, the hydroxyl radical would appear to be generated and to react with the constituents of the cream to produce a new radical or “secondary radical”, observed in the form of adduct on the DIPPMPO.
[0152] In conclusion, the colloids of the invention have capacity greater than the other compounds tested to trap free radicals of the superoxide type. Also, no production of secondary radical species was observed with the colloids of the invention, showing total effectiveness on inhibiting ROS and thus on inhibiting oxidative stress.